Summary of work: In obesity-related insulin resistance, chronic exposure to elevated concentrations of glucose can alter the thiol redox state of the cells by a mechanism that involves generation of reactive oxygen species. The insulin receptor contains reactive sulfhydryl group(s) in the cytoplasmic domain of the receptor beta-subunit whose function remain unclear. Changes in reactivity of this receptor thiol group(s) has been evaluated with a thiol-biotinylating agent. Cells were incubated in the presence of 5.7 mM or 25 mM glucose for varying amounts of time. Also, the effect of hydrogen peroxide alone or together with vanadate was assessed. Immunoprecipitation of the insulin receptor followed by immunoblotting with streptavidin-bound agarose has indicated a time-dependent reduction in receptor thiol reactivity by high glucose. Vanadate alone did not alter the reactivity of IR thiol(s) but greatly enhanced the oxidative effect of hydrogen peroxide. These results indicate that there is a critical thiol group(s) in the cytoplasmic domain of the insulin receptor that is susceptible to glucose and intracellular oxidants. This is the first evidence for alteration of receptor thiol reactivity under conditions that involve generation of low levels of reactive oxygen species, which suggests that this alteration might regulate the receptor function and/or its mode of interaction with other cellular constituents. Specific inhibitors of several oxidant-generating enzymes, including cyclo-oxygenase, cytochrome P-450, nitric oxide synthase, NADPH oxidase, xanthine oxidase, and lipoxygenase are being used to assess the function of these enzymes in glucose-mediated reduction in receptor thiol reactivity and signal transduction. Progress is being made in the identification of the reactive receptor thiol group(s).